The invention relates to a disposable or reusable device for carrying out a process in which a biological sample is processed with one or more reagents. The invention relates in particular to a device which is suitable for carrying out a process for obtaining a purified nucleic acid sample from a biological sample.
Known methods for obtaining a purified nucleic acid sample suitable to be amplified, by, for example, a polymerase chain reaction (PCR) are usually carried out manually and involve a number of steps and in particular a plurality of pipetting operations. Since contamination of the purified sample to be obtained has to be reduced as far as possible, the manual process has to be carried out with great care and is therefore a time consuming task. Known apparatus for automatically carrying out pipetting operations in analyzer systems have been found inadequate for methods aiming to obtain purified nucleic acid samples suitable to be amplified, such as by a polymerase chain reaction (PCR), because contamination of the sample is likely to occur during pipetting operations.
Amplification reactions allow the detection of minute amounts of nucleic acids. When amplifications are carried out according to the PCR principle (e.g. U.S. Pat. No. 4,683,195), the samples must be prepared in a particularly careful manner in order to isolate the nucleic acids as there are many biological substances which may interfere with the function of the polymerase used in this method. Due to the extremely high sensitivity of this method, it is also necessary that carry-over be avoided in this procedure under all circumstances.
It is well known that the PCR is carried out in a multi-well plastic plate which contains a standard number of wells, i.e., 96 or 384. The small volumes of the wells are generally in the range 30 and 100 millimicroliters, a volume range for water based solutions where even a short exposure to non-equilibrium water vapor air and/or substantial turbulence causing gas volume exchange above the liquid in the wells results in substantial volume and/or non-water component damage as a result of turbulence, condensation and/or evaporation effects. This applies in particular if frequent changing of the temperature of the solutions between high and low temperatures is required, as occurs in the case of the PCR, wherein individual nucleic acid strands are susceptible to undesirable fragmentation, concentration or dilution. The high degree of sensitivity of these tests translates into a high degree of required care with the super-liquid surface gas above the sample surface in the well.
It is clear that the well liquid must have a cover with a gas tight seal. The prior art solves this problem in many ways. The problem with these prior art seals for a multi-well plate is that the seals are either joined in a continuous sheet such that the removal and reapplication after thermal cycling causes condensate to cling to the inside seal surface so that substantial amounts of the sample liquid are dried quickly or drop into other wells or the seals are individual plugs which are removed via single removal or require a special interface to remove or replace the plugs. There is clearly a need for a continuous sheet of well seals that eliminates liquid loss and contamination as experienced in the prior art.
The invention comprises a wide range of pierceable and memory-recloseable septa cover for multi-well plates for PCR or other small sample well processes, where such seals are joined into an easily applied or removed sheet or otherwise joined means. A reaction chamber well comprises a liquid level portion at the bottom that contains the various levels of reaction liquid, a super-liquid portion comprising the gas volumes above the reaction liquid. In one form of the invention embodiments, the cover comprises a gas tight seal between the cover and the side of the well substantially above the liquid level, thereby forming the super-liquid space. The disturbance, pressure change and/or gas exchange in this small gas space results in a rather dramatic potential change in reaction or mixing results. The invention cover reduces each of these to a minimum not attainable in the prior art.
The invention cover in one embodiment comprises a concave or hollow cylindrical (i.e., convex or flat as to the super-liquid space) user side that is slightly pressed on an outer surface against the side of the rigid well inner walls. At the bottom of the concavity or hollow cylindrical portion is a beveled or rounded impression having along its lowest impression trace a minimum thickness defining the pierceable septum, pierceable by the standard pipette tip. A most critical aspect of the invention is the presence of the impression whereby the septum opening and closing within the super-liquid space comprises a minimum disturbance into that space.
An example of the PCR follows as typical of liquid addition and/or subtraction by pipette tip or other relatively rigid cannula or conduit with an open end, which is also referred to as just a tip.
The invention seals may be utilized to seal a small well reaction chamber for a polynucleotide amplification reaction. The reaction chamber may be provided with reagents for PCR including a sample polynucleotide, polymerase, nucleoside triphosphates, a first primer hybridizable with the sample polynucleotide, and a second primer hybridizable with a sequence that is complementary to the sample polynucleotide, wherein the first and second primers define the termini of the amplified polynucleotide product. The well and seal will experience thermal cycling the contents of the amplification reaction chamber, such that, in each cycle, e.g., the temperature is controlled to 1) dehybridize (xe2x80x9cmeltxe2x80x9d) double stranded polynucleotide, 2) anneal the primers to single stranded polynucleotide, and 3) synthesize amplified polynucleotide between the primers. Other amplification methods available in the art also may be utilized, including, but not limited to: (1) target polynucleotide amplification methods such as self-sustained sequence replication (3SR) and strand-displacement amplification (SDA); (2) methods based on amplification of a signal attached to the target DNA, such as xe2x80x9cbranched chainxe2x80x9d DNA amplification (Chiron Corp.); (3) methods based on amplification of probe DNA, such as ligase chain reaction (LCR) and QB replicase amplification (QBR); and (4) various other methods such as ligation activated transcription (LAT), nucleic acid sequence-based amplification (NASBA), repair chain reaction (RCR) and cycling probe reaction (CPR) (for a review of these methods, see pp. 2-7 of The Genesis Report, DX, Vol. 3, No. 4, February 1994; Genesis Group, Montclair, N.J.).
A main object of the invention seal is to provide an assembly to ensure a contamination-free processing of samples and reagents to a degree which is sufficient to comply with the requirements of nucleic acid purification methods which provide nucleic acid samples having a high degree of purity and being thereby suitable to be amplified.